Some strains of Theiler's murine encephalitis virus (TMEV), a picornavirus, cause demyelinating disease in mice which is a model system for diseases such as multiple sclerosis. The recent availability of cDNA clones of BeAn, a strain of TMEV which produces demyelination in SJL and other strains of mice, and GDVII, a strain which cause acute encephalitic infections, allows these to be used to study individual viral proteins, both for their function and for their role in immunity. The first major aim of the proposed research is to express the capsid protein region of BeAn in vaccinia virus vectors, using either the intact capsid region or regions representative of the individual proteins, VP1 and VP3. In studies in which the intact capsid region is to be studied, a vaccinia derivative which expressed the viral protease responsible for cleavage of the protein precursor of the capsid proteins will be used. Co-infection of cells with viruses which encode the capsid region and the protease should allow production of the individual proteins and, possibly, assembly of capsid intermediates. The vaccinia recombinants will be used to induce immunity in mice. For in vitro studies, cells infected with the recombinants will be used to study cytotoxic T cell reactions, in collaboration with Dr. C. Waltenbaugh. All constructs being made also allow in vitro transcription and translation of viral proteins. This approach will be used to make antigens for studies of delayed-type hypersensitivity reactions in vitro (class Il- restricted proliferation responses). Furthermore, genetic manipulation of the plasmids will be used to further define regions of the individual proteins recognized by different immune cells. In a second set of experiments, initial studies of TMEV non- structural proteins will be undertaken. Focus will be on the viral leader polypeptide, a protein of unknown function in picornaviruses which encode it, and on the 2A/2B protein, which spans a region used in cleavage of the primary polyprotein of TMEV. The 2A/2B region will be expressed in bacteria to determine whether autocatalytic cleavage occurs or in vitro to determine whether cleavage requires the presence of host translational machinery. Once cleavage is demonstrated, derived mutants will be used to define necessary sequences for this proteolytic reaction.